Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration

“…In our lab, GbpA has routinely been produced in E. coli , in both TB and minimal media, and purified from the E. coli periplasmic fraction by ion-exchange chromato­graphy and SEC. Interestingly, we found in previous work that better yields were obtained when producing the protein in minimal media instead of the nutrient-rich media TB (15 mg per L in M9 vs 7 mg per L in TB) . We suspected that this might be due to misfolding of GbpA when produced too quickly in TB, whereas slower expression in M9 would result in less aggregates.…”

“…This adhesin binds to chitin and mucins, helping the pathogen to survive in its natural marine environment and to colonize the human intestine, respectively . In our lab, GbpA has routinely been produced in E. coli , in both TB and minimal media, and purified from the E. coli periplasmic fraction by ion-exchange chromato­graphy and SEC. Interestingly, we found in previous work that better yields were obtained when producing the protein in minimal media instead of the nutrient-rich media TB (15 mg per L in M9 vs 7 mg per L in TB) .…”

Tangled up in fibers: How a lytic polysaccharide monooxygenase binds its chitin substrate

“…Perdeuteration of GbpA involved expression in deuterated media, according to the protocol described in [15], which was inspired by [16]. This protocol relies on BL21 star (DE3) cells containing the GbpA-encoding gene in the pET22b vector [15]. Growth and expression media contained D2O and d8-glycerol (99% D) purchased from ChemSupport AS (Hommelvik, Norway).…”

“…Growth and expression media contained D2O and d8-glycerol (99% D) purchased from ChemSupport AS (Hommelvik, Norway). The production of H-GbpA and the purification of D-GbpA and H-GbpA is also described in [15].…”

“…The structure of the first three domains of GbpA has been solved by X-ray crystallography [12], recently complemented with the full-length crystal structure of a GbpA homolog from Vibrio campbellii [14]. In addition, the solution structure of GbpA has been characterized with Small-Angle X-ray Scattering (SAXS) and Small-Angle X-ray Scattering (SANS) [12,15], revealing a monomeric elongated structure. How the structure of GbpA changes upon binding to chitin, and how the domains align in this state, is, however, unknown.…”